Hemoglobin, a protein containing iron in a protoporphyrin IX prosthetic group, functions physiologically as the principal carrier of oxygen in whole blood from the lungs to other body tissues. It is also the protein found in highest concentration in whole blood (normally 12-18 gram percent). Lower than normal values are symptomatic of anemia. Higher than normal values are indicative of polycythemia or erythrocytosis. The determination of hemoglobin content of whole blood is done routinely and, thus, is one of the most frequently performed clinical laboratory tests.
Numerous methods and devices for the determination of hemoglobin are known. Hemoglobin can be assayed directly, as in the Tallquist Method, by measuring the transmission or reflection optical density of the red color imparted by oxyhemoglobin (one form of hemoglobin) without any further chemical modification of the hemoglobin. Such "direct" measurement is described, for example, in U.S. Pat. Nos. 4,057,394 (issued Nov. 8, 1977 to Genshaw) and 4,337,222 (issued June 29, 1982 to Kitajima et al).
However, hemoglobin exists in blood in several forms [e.g. oxyhemoglobin (HbO.sub.2), deoxyhemoglobin (Hb), methemoglobin (metHb), carboxyhemoglobin (HbCO) and sulfhemoglobin (HbS)], each with a unique absorption spectrum. See, e.g. Fundamentals to Clinical Chemistry, N. W. Tietz (Ed.), W. B. Saunders, Co., Philadelphia, 1970, pp. 263-267.
It would be desirable to measure all forms of hemoglobin to determine total hemoglobin with a single measurement. This requires the chemical conversion of the various forms of hemoglobin into a single detectable and stable form. Attempts have been made using "indirect" assay methods whereby most forms of hemoglobin are converted to one detectable end product. In one commonly used method, the iron in hemoglobin is oxidized (from Fe.sup.+2 to Fe.sup.+3) with a ferricyanide, thus converting the hemoglobin forms to methemoglobin, followed by conversion of the methemoglobin to cyanmethemoglobin with a cyanide. This well known Drabkin's method is fairly rapid and suitable for solution assay. It has been adopted internationally as the approved standard method for hemoglobin measurement (see J. Clin. Path., 31, pp. 139-143 (1978)).
Dry chemistry assays are known. Such assays are analytical techniques wherein chemical reagents are incorporated in various substantially "dry-to-the-touch" elements, e.g. test strips and multizone analytical elements. The advantages of dry chemistry assays over wet chemistry assays (i.e. techniques using reagents in solutions) are also known and include simplicity of use, economic savings and rapid analysis. See, for example, U.S. Pat. No. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al).
However, in attempting to adapt the indirect Drabkin method to existing dry chemistry assays, several problems have been encountered. In solution assays, a 250-fold dilution of the blood sample is necessary. However, such dilution is undesirable for automated analyses because it complicates the analyses. Hence, the concentration of hemoglobin would be significantly higher in undiluted dry assays and the small amount of Drabkin's reagent (i.e. cyanide) generally used in diluted solution assays is insufficient to provide an accurate dry assay. To compensate for this deficiency in a dry element, the amount of ferricyanide and cyanide must be significantly increased. However, potassium cyanide, which is commonly used in the Drabkin test, is extremely toxic particularly at the higher concentrations. The danger presented to those who would prepare or use dry analytical elements containing high amounts of potassium cyanide is unacceptable. Hence, it would be desirable to replace this particular reagent while obtaining highly quantitative analyses.
Other agents are known to react with the oxidized forms of hemoglobin. For example, Perutz et al in Biochem., 17, pp. 3640-3652 (1978), describe a study of the reaction of various forms of hemoglobin with thiocyanate. No hemoglobin assay using dry elements is described in this reference.
It has also been observed that some surface active agents (i.e. surfactants) can convert various forms of hemoglobin into detectable products. For example, U.K. patent No. 2,052,056 (published Jan. 21, 1981) describes the use of water-soluble nonionic detergents in alkaline solution to convert hemoglobin and its derivatives to an end product measurable at 575 nm. A similar use of anionic or cationic detergents is particularly discouraged in this reference's teaching. Another reference, namely Oshiro et al, Rinsho Byorei, 29(2), pp. 203-209 (1981) describes the use of sodium dodecyl sulfate in a solution assay for hemoglobin.
However, attempts to adapt these known solution assay techniques to dry chemistry assays have not provided acceptable results. Although dry elements for hemoglobin assays have previously been prepared containing a thiocyanate or anionic surfactant reagent, those elements exhibited poor precision, that is, the random error in analytical measurements has been unacceptably high when they were used.
Hence, there is a need in the art for a simple dry chemistry element for rapid determination of hemoglobin in whole blood, which assay avoids toxic reagents, exhibits improved precision and obviates the need to wipe off excess blood.